Introduction
As at-risk populations grow, invasive fungal infections keep rising. The high morbidity and mortality rates associated with fungal diseases require ongoing research into novel antifungal agents and diagnostic techniques using substitute biomarkers. The use of biologic disease markers as prognostic indicators in clinical care may speed up the completion of clinical trials by requiring fewer participants, incurring lower trial costs, and necessitating less extensive long-term follow-up than would otherwise be the case. Numerous fungi-related biomarkers have been created and thoroughly tested in small series and prospective clinical trials. In this review, we look at the supporting data for these substitute biomarkers and offer suggestions for regulators and medical professionals.
Cryptococcosis
Surrogate biomarkers for cryptococcosis can be used for diagnosis, prognosis, and treatment decisions. Standard diagnostics are India ink microscopy, cryptococcal antigen (CrAg), and culture. Culture and microscopy techniques date to 1894 and are still used. India ink testing for visualization of the capsule can detect ~103 CFU yeasts/mL in centrifugated cerebrospinal fluid (CSF) and has a sensitivity of ~60%–80% in HIV-infected patients and ~50% in non-HIV cryptococcal meningitis. The change in quantitative CSF microscopy yeast counts correlates poorly with changes in quantitative culture and cannot be used as a surrogate marker. CrAg is an established diagnostic biomarker that is more sensitive than culture. CrAg LFA is most sensitive. CrAg is preferred over BDG or PCR testing for diagnosis, but sequential CSF CrAg titers should not be used for monitoring response to therapy. The CSF clearance rate of Cryptococcus from serial quantitative CSF cultures (EFA) is a direct marker of antifungal activity and a surrogate biomarker of mortality and is now routinely used to evaluate novel antifungal strategies in cryptococcal meningitis clinical trials.
Invasive candidiasis
Invasive candidiasis includes bloodstream infections by Candida spp. (candidemia), which may be complicated by end-organ infections from hematogenous dissemination, and noncandidemic infections of organs or submucosal tissues. Data are most robust for 3 culture-independent diagnostic tests: (1->3)-β–D-glucan, Candida polymerase chain reaction (PCR), and T2Candida assays of blood-based samples. Culture-independent diagnostic test–guided patient management strategies have not been validated in clinical trials. Cultures remain the mainstay of candidiasis diagnostics yet should not be the limiting inclusion criterion for invasive candidiasis trials. Documented clearance of candidemia is a marker of successful antifungal treatment. We recommend using diagnostic molecular assays on positive cultures to accurately and rapidly identify organisms to species level. Culture-independent diagnostic tests may be useful in selected clinical scenarios to initiate or de-escalate antifungal therapy. Positive PCR or T2Candida testing in patients with risk factors for candidiasis warrants enrollment in clinical trials. Multiple possible biomarkers exist that could be used in phase II trials to quantify antifungal activity, but these biomarkers have not been validated.
Aspergillosis
Aspergillus spp. are ubiquitous in the environment and frequently inhaled into the airways . Diagnosis of invasive aspergillosis requires an appropriate host and compatible clinical syndrome when cultures from the airway return positive to differentiate colonization from infection. The Aspergillus cell wall contains polysaccharides, BDG, chitin, and other components that may be utilized for detection in patient samples. Galactomannan, BDG, and PCR are standard diagnostic tests used in clinical practice, and some PCR assays have the advantage of also detecting mutations conveying antifungal drug resistance. BDG has the least specificity and is insufficient as a diagnostic biomarker for enrollment of patients into clinical trials. For prognostic markers of response to antifungals, the change in blood galactomannan values over time is a promising serial surrogate marker. PCR negativity over time is also promising for use as a surrogate marker of patient outcomes.
Other molds
Diagnostics for non-Aspergillus molds require further development, and specific biomarkers for these organisms would be helpful in disease management. Insufficient data exist on the utility of using any surrogate endpoints in clinical trials.
Blastomycosis
Endpoints for successful outcomes of treatment in blastomycosis classically have been clinical improvement together with the resolution of skin lesions or radiologic findings. A standard assay for the diagnosis of probable blastomycosis is a Blastomyces antigen, which measures the cell wall polysaccharide (galactomannan) of Blastomyces dermatitidis in urine, serum, or body fluids. However, there are insufficient data to recommend biomarkers for the prognosis of blastomycosis or as surrogate endpoints in clinical trials. Longitudinal change in serum Blastomyces antigen requires further investigation. Surrogate markers for the prognosis of the more severe disease manifestations of acute respiratory distress syndrome in blastomycosis are needed.
Coccidioidomycosis
Past studies of coccidioidomycosis have used combined clinical scores, radiologic results, and serologic results to determine a composite score for clinical severity. Serologic testing may be performed using a number of methodologies. Serologic testing is useful for the diagnosis of coccidioidomycosis, and EIA results should be confirmed by immunodiffusion. Longitudinal assessment using CF serology is useful to monitor the response to antifungal therapy. However, insufficient data exist on their utility as surrogate end points for use in clinical trials.
Histoplasmosis
Historically, the first prognostic marker for histoplasmosis was duration of positive blood cultures in patients with HIV who were receiving antifungal therapy. In studies performed during the early AIDS era, recurrence of disease was associated with positive or persistently positive blood culture. The most widely used surrogate marker for histoplasmosis diagnosis is the Histoplasma polysaccharide galactomannan antigen, which can be detected in urine or serum. Urine Histoplasma antigen is well studied in patients with HIV and is useful in diagnosis, patient management, and as a potential endpoint in clinical trials. In other populations, further studies should be performed to define the usefulness of longitudinal antigen testing to determine prognosis.
Paracoccidioidomycosis
Serologic testing is frequently used in the diagnosis of paracoccidioidomycosis and is highly specific (>95%) and sensitive (~80%). Numerous methodologies have been investigated, including in-house assays, although a commercial immunodiffusion test is now available. A previous study found longitudinal serologic assessment useful to monitor response to therapy. However, insufficient data exist on serology as a surrogate endpoint for use in clinical trials.
Sporotrichosis
Several assays are in development that may offer promise in the diagnosis of sporotrichosis, and they may be useful biomarkers during future clinical trials following validation. Currently, there are no biomarkers that can be used as a surrogate end point in clinical trials.
Talaromycosis
Culture remains the mainstay of diagnosis for talaromycosis. Antigen testing may be useful in future clinical trials following validation, but currently cannot be recommended as a surrogate end point in clinical trials. The longitudinal change in quantitative blood CFUs has been found useful to quantify the EFA in a recent clinical trial and should be a standard metric in future clinical trials.
Future directions
The development of affordable, readily available, and rapid in-house diagnostic tests is critically important. Cryptococcosis and talaromycosis are the only fungal infections with established surrogate endpoints, and further investigation of surrogate markers for other invasive fungal infections such as invasive candidiasis and aspergillosis is urgently needed to aid in both diagnosis and prognosis.
Source: Source – George R Thompson, III, David R Boulware, Nathan C Bahr, Cornelius J Clancy, Thomas S Harrison, Carol A Kauffman, Thuy Le, Marisa H Miceli, Eleftherios Mylonakis, M Hong Nguyen, Luis Ostrosky Zeichner, Thomas F Patterson, John R Perfect, Andrej Spec, Dimitrios P Kontoyiannis, Peter G Pappas, Noninvasive Testing and Surrogate Markers in Invasive Fungal Diseases, Open Forum Infectious Diseases, Volume 9, Issue 6, June 2022, ofac112, https://doi.org/10.1093/ofid/ofac112.
